The overall goal of this project is the discovery of new natural products with anticancer activity from extremophilic microbes isolated from an acid mine waste lake. Compounds will be isolated based on their ability to inhibit specific signal transduction enzymes that have been implicated in the onset and metastasis of several different types of cancer. Matrix metalloproteinase-3 (MMP-3) and caspase-1 (casp-1) have been shown to be up-regulated in certain cancers. For the past five years we have developed a bioassay-guided isolation scheme that has resulted in the isolation and purification of several novel compounds that inhibit one or both of these enzymes. The structures of these compounds were elucidated through spectral techniques. To establish a correlation between enzyme inhibition and anticancer activity, these pure enzyme inhibitors were submitted to the National Cancer Institute Developmental Therapy Program (NCI/DTP) for testing against 60 human cancer cell lines. This methodology has resulted in the isolation and identification of several lead compounds with nanomolar or low micromolar activity and high selectivity towards ovarian cancer, non-small cell lung cancer, and leukemia. Microbes isolated from acid mine waste have proven to be a rich source of previously unreported, bioactive metabolites. Preliminary studies, which have been funded through the National Institutes of Health-IDeA Networks of Biomedical Research Excellence (INBRE), yielded over 70 different microorganisms from the metal sulfate rich waters and deep basal sediment. Of the 30 microorganisms already examined in small pilot fermentation studies, over 50% of the extracts inhibited MMP-3 and/or Casp-1. In- depth exploration of five of these organisms has yielded several novel compounds including berkelic acid, which exhibited selective nanomolar activity against OVCAR-3 and berkeleydione which exhibited selective low micromolar activity against nonsmall cell lung cancer strain NCI-H460. With these early successes we would like to continue studying these microbes with the following specific aims: 1. Isolate compounds from targeted extremophilic microorganisms using signal transduction enzyme inhibition as an isolation guide. 2. Elucidate the structures of these compounds using spectroscopic or x-ray methodology. 3. Test active compounds in the NIH/DTP cell line screen to determine in vitro activity. 4. Send compounds with enzyme inhibitory activity and nanomolar - low micromolar activity against specific human cancer cell lines to Eisai Research Institute for further evaluation. 5. Involve undergraduate students in all aspects of microbial drug discovery This research has shown promise and the funds requested for this RO1 would be used to continue this exploration. It addresses a critical need area and combines both unique assay tools and a unique extreme environment for drug discovery. PUBLIC HEALTH RELEVANCE: Over 565,000 Americans are expected to die of cancer this year, and more than 2,437,000 new cancer cases are expected to be diagnosed. Despite our arsenal of anticancer agents, cancer is now the second leading cause of death in the western world. Unfortunately, current treatment modalities use cytotoxic agents that can cause deleterious side-effects. This application describes a novel approach to drug discovery, using signal transduction enzyme inhibition as a guide to the isolation of selective, potent anticancer agents from extremophilic microbes. Enzyme inhibitors will be tested by NCI/DTP against 60 human cancer cell lines and by Eisai Research Institute to determine anticancer activity. Novel compounds will help meet the increasing need for new treatment modalities with high selectivity and potency, and lower cytotoxicity than current treatment options.